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The FASEB Journal • Research Communication
Methionine restriction decreases mitochondrial oxygen
radical generation and leak as well as oxidative
damage to mitochondrial DNA and proteins
Alberto Sanz,* Pilar Caro,* Victoria Ayala,
†
Manuel Portero-Otin,
†
Reinald Pamplona,
†
and Gustavo Barja*
,1
*Department of Animal Physiology-II, Complutense University, Madrid, Spain;
†
Department of Basic
Medical Sciences, University of Lleida, Lleida, Spain
ABSTRACT Previous studies have consistently shown
that caloric restriction (CR) decreases mitochondrial
reactive oxygen species (ROS) (mitROS) generation
and oxidative damage to mtDNA and mitochondrial
proteins, and increases maximum longevity, although
the mechanisms responsible for this are unknown. We
recently found that protein restriction (PR) also pro-
duces these changes independent of energy restriction.
Various facts link methionine to aging, and methionine
restriction (MetR) without energy restriction increases,
like CR, maximum longevity. We have thus hypothe-
sized that MetR is responsible for the decrease in
mitROS generation and oxidative stress in PR and CR.
In this investigation we subjected male rats to exactly
the same dietary protocol of MetR that is known to
increase their longevity. We have found, for the first
time, that MetR profoundly decreases mitROS produc-
tion, decreases oxidative damage to mtDNA, lowers
membrane unsaturation, and decreases all five markers
of protein oxidation measured in rat heart and liver
mitochondria. The concentration of complexes I and
IV also decreases in MetR. The decrease in mitROS
generation occurs in complexes I and III in liver and in
complex I in heart mitochondria, and is due to an
increase in efficiency of the respiratory chain in avoid-
ing electron leak to oxygen. These changes are strik-
ingly similar to those observed in CR and PR, suggest-
ing that the decrease in methionine ingestion is
responsible for the decrease in mitochondrial ROS
production and oxidative stress, and possibly part of
the decrease in aging rate, occurring during caloric
restriction.—Sanz, A., Caro, P., Ayala, V., Portero-Otin,
M., Pamplona, R., Barja, G. Methionine restriction
decreases mitochondrial oxygen radical generation and
leak as well as oxidative damage to mitochondrial DNA
and proteins. FASEB J. 20, 1064–1073 (2006)
Key Words: mitochondria 䡠 methionine restriction 䡠 caloric re-
striction 䡠 free radicals 䡠 aging 䡠 DNA damage 䡠 oxidative
damage
The results of many experimental and comparative
studies are consistent with the validity of the mitochon-
drial oxygen radical theory of aging (1,2). These studies
suggest that the mitochondrial rate of generation of
ROS (mitROS generation) plays a main role. Many
investigations have shown that mitROS generation is
lower in long-lived than in short-lived animal species
and that caloric restriction (CR) consistently decreases
the rate of mitROS production (reviewed in ref 3). In
these two models, low rates of mitROS generation are
accompanied by lower levels of oxidative damage to
mitochondrial DNA (mtDNA) and proteins (2,4–6).
Although numerous studies have documented the
decrease in MitROS production and oxidative damage
to mitochondrial macromolecules in CR, the dietary
factor that causes these beneficial changes is unknown.
Recent studies are beginning to clarify this subject.
Thus, while lipid restriction does not change mitROS
production (7), we recently found that protein restric-
tion decreases mitROS production in rat liver (8)
independent of energy intake, although the protein
component responsible for this is not known. This
decrease occurs specifically at complex I, lowers the
percent free radical leak (FRL) in the respiratory chain,
and decreases oxidative damage to mtDNA in rat liver
(8). All these changes also occur in CR with a similar
magnitude, suggesting that restriction of protein intake
can be responsible for the well-known decreases in
mitROS production and oxidative stress that take place
in CR. On the other hand, although it has been
believed that the antiaging effect of CR is due to the
decreased intake of calories themselves rather than to
decreases in specific dietary components, recent find-
ings question this consensus (9). Moreover, classic
studies have repeatedly found that protein restriction
increases maximum longevity in rats and mice (10–13),
although the magnitude of this increases are around
half that of CR. In addition, it has been observed that
methionine restriction (MetR) without energy restric-
tion increases maximum longevity in rats and mice
1
Correspondence: Departamento de Fisiologı´a Animal-II,
Facultad de Ciencias Biolo´gicas, Universidad Complutense,
c/Antonio Novais-2, Madrid 28040, Spain. E-mail: gbarja@
bio.ucm.es
doi: 10.1096/fj.05–5568com
1064 0892-6638/06/0020-1064 © FASEB
(14,15). Various other kinds of recent investigations
also point to a relationship between methionine and
longevity (16–19).
Taking into account all these findings, we hypothe-
sized that the restriction in methionine intake can be
the cause of the decrease in mitROS generation and
oxidative stress observed in CR and in protein restric-
tion in rodents, and so can be responsible for around
half of the increase in maximum longevity observed in
caloric restriction. To test that hypothesis, in the
present investigation we subjected male rats to exactly
the same dietary protocol of MetR that is known to
increase rat maximum longevity (14). The time of
restriction used (6 –7 wk) was the same that induces
decreases in mitROS generation in the liver of rats
subjected to both caloric restriction (20) and protein
restriction (8). In liver and heart mitochondria of these
methionine-restricted rats, we measured the rates of
mitROS generation, mitochondrial oxygen consump-
tion, FRL, the concentration of complexes I and IV, five
oxidative damage markers of protein oxidation (the
specific protein carbonyls glutamic and aminoadipic
semialdehydes, GSA and AASA), glycoxidation (car-
boxyethyl-lysine CEL and carboxymethyl-lysine CML)
and lipoxidation (malondialdehydelysine MDAL, and
CML), as well as oxidative damage to mitochondrial
DNA. Since it is known that the degree of unsaturation
of phospholipids can affect markers of protein lipoxi-
dation, the full fatty acid composition was measured in
liver and heart mitochondria of methionine-restricted
and control animals.
MATERIALS AND METHODS
Animals and diets
Male Wistar rats of 250 g of body wt were obtained from
Iffa-Creddo (Lyon, France). The animals were caged individ-
ually and maintained in a 12:12 (light:dark) cycle at 22 ⫾ 2°C
and 50 ⫾ 10% relative humidity. The semipurified diets used
in this investigation were prepared by MP Biochemicals
(formerly ICN), Irvine, CA, USA. Control animals were fed ad
libitum a semipurified diet based on the American Institute of
Nutrition AIN-93G diet: 31.80% cornstarch, 31.80% sucrose,
5.00% dextrin, 8.00% corn oil, 5.00% alphacel non-nutritive
bulk, 1.12% l-arginine, 1.44% l-lysine, 0.33% l-hystidine,
1.11% l-leucine, 0.82% l-isoleucine, 0.82% l-valine, 0.82%
l-threonine, 0.18% l-tryptophan, 0.86% l-methionine, 2.70%
l-glutamic acid, 1.16% l-phenylalanine, 2.33% glycine, 0.20%
choline bitartrate, 1.00% AIN vitamin mix, and 3.50% AIN
mineral mix. The methionine-restricted rats received the
same diet except that l-methionine was 0.17% and l-glutamic
acid was 3.39%. Control animals received daily the same
amount of food that the methionine-restricted animals had
eaten as a mean during the previous day (pair feeding). Using
this diet and protocol, it has been demonstrated that methi-
onine restriction increases maximum longevity in rats (14).
After 6 –7 wk of dietary treatment the animals were sacrificed
by decapitation. The liver and heart were immediately pro-
cessed to isolate mitochondria, and liver and heart samples
were stored at – 80°C for the assays of oxidative damage to
mtDNA.
Mitochondria isolation
Rat liver and heart mitochondria were obtained from fresh
tissue. The liver was rinsed and homogenized in 60 ml of
isolation buffer (210 mM mannitol, 70 mM sucrose, 5 mM
HEPES, 1 mM EDTA, pH 7.35). The nuclei and cell debris
were removed by centrifugation at 1000 g for 10 min. Super-
natants were centrifuged at 10,000 g for 10 min and the
resulting supernatants were eliminated. The pellets were
resuspended in 40 ml of isolation buffer without EDTA and
centrifuged at 1000 g for 5 min. Mitochondria were obtained
after centrifugation of the supernatants at 10,000 g for 10
min. After each centrifugation step, any overlaying layer of fat
was eliminated. Heart mitochondria were isolated by differ-
ential centrifugation as described previously (4) except that
the centrifugations were performed at 1000 g for 10 min to
eliminate nuclei and cellular debris and at 10,000 g to pellet
mitochondria. The mitochondrial pellets were resuspended
in 1 ml of isolation buffer without EDTA. All the above
procedures were performed at 5°C. Mitochondrial protein
was measured by the Biuret method. The final mitochondrial
suspensions were maintained over ice and used immediately
for measurements of oxygen consumption and H
2
O
2
produc
-
tion.
Mitochondrial H
2
O
2
production
The rate of mitochondrial H
2
O
2
production was assayed by
measuring the increase in fluorescence (excitation at 312 nM,
emission at 420 nM) due to oxidation of homovanillic acid by
H
2
O
2
in the presence of horseradish peroxidase (21). Reac
-
tion conditions were 0.25 mg of mitochondrial protein per
ml, 6U/ml of horseradish peroxidase, 0.1 mM homovanillic
acid, 50 U/ml of superoxide dismutase (SOD), and 2.5 mM
pyruvate/2.5 mM malate, 2.5 mM glutamate/2.5 mM malate,
or 5 mM succinate ⫹ 2 M rotenone as substrates, added at
the end to start the reaction to the incubation buffer (145
mM KCl, 30 mM HEPES, 5 mM KH
2
PO
4
, 3 mM MgCl
2
, 0.1
mM EGTA, 0.1% albumin, pH 7.4) at 37°C, in a total vol of
1.5 ml. Unless otherwise stated, the assays with succinate as
substrate were performed in the presence of rotenone in
order to avoid the backward flow of electrons to Complex I.
In some experiments rotenone (2 M) or antimycin A (2
M) were also included in the reaction mixture to assay
maximum rates of Complex I or Complex III H
2
O
2
genera
-
tion. Duplicated samples were incubated for 15 min at 37°C;
the reaction was stopped by transferring the samples to a cold
bath and adding 0.5 ml of stop solution (2.0 M glycine, 2.2M
NaOH, 50 mM EDTA), and the fluorescence was read in a
LS50B Perkin-Elmer fluorometer. Known amounts of H
2
O
2
generated in parallel by glucose (Glc) oxidase with Glc as
substrate were used as standards. Since the SOD added in
excess converts all O
2
.–
excreted by mitochondria (if any) to
H
2
O
2
, the measurements represent the total (O
2
.–
plus
H
2
O
2
) rate of mitochondrial ROS production.
Mitochondrial oxygen consumption
The oxygen consumption of liver and heart mitochondria was
measured at 37°C in a water-thermostatized incubation cham-
ber with a computer-controlled Clark-type O
2
electrode (Oxy
-
graph, Hansatech, UK) in 0.5 ml of the same incubation
buffer used for H
2
O
2
measurements. The substrates used
were complex I- (2.5 mM pyruvate/2.5 mM malate or 2.5 mM
glutamate/2.5 mM malate) or complex II-linked (5 mM
succinate⫹2 M rotenone). The assays were performed in
the absence (State 4, resting) and in the presence (State 3,
phosphorylating) of 500 M ADP.
1065METHIONINE RESTRICTION AND ROS GENERATION
Mitochondrial free radical leak
The H
2
O
2
production and O
2
consumption of liver and heart
mitochondria were measured in parallel in the same samples
under similar experimental conditions. This allowed the
calculation of the fraction of electrons out of sequence, which
reduce O
2
to ROS at the respiratory chain (the percent free
radical leak, FRL) instead of reaching cytochrome oxidase to
reduce O
2
to water. Since two electrons are needed to reduce
1 mol of O
2
to H
2
O
2
whereas four electrons are transferred in
the reduction of 1 mol of O
2
to water, the percent free radical
leak was calculated as the rate of H
2
O
2
production divided by
twice the rate of O
2
consumption, and the result was multi
-
plied by 100.
Measurement of 8-oxodG in mtDNA
Mitochondrial DNA (mtDNA) was isolated by the method of
Latorre et al. (22) adapted to mammals (23). The isolated
nuclear and mitochondrial DNAs were digested to de-
oxynucleoside level by incubation at 37°C with5Uof
nuclease P1 (in 20 l of 20 mM sodium acetate, 10 mM ZnCl
2
,
15% glycerol, pH 4.8) for 30 min and1Uofalkaline
phosphatase (in 20 l of 1 M Tris-HCl, pH 8.0) for 1 h (24).
All aqueous solutions used for mtDNA isolation, digestion,
and chromatographic separation were prepared in HPLC-
grade water. Steady-state oxidative damage to mtDNA was
estimated by measuring the concentration of 8-oxodG, re-
ferred to that of the nonoxidized base (deoxyguanosine, dG).
8-oxodG and dG were analyzed by HPLC with on-line elec-
trochemical and UV detection, respectively. The nucleoside
mixture was injected into a reverse-phase Beckman Ultra-
sphere ODS column (5 m, 4.6 mM⫻25 cm) and was eluted
with a mobile phase containing 2.5% acetonitrile and 50 mM
phosphate buffer pH 5.0. A Waters 510 pump at 1 l/min was
used. 8-oxodG was detected with an ESA Coulochem II
electrochemical coulometric detector (ESA, Inc. Bedford,
MA, USA) with a 5011 analytical cell run in the oxidative
mode (225 mV/20 nA), and dG was detected with a Bio-Rad
model 1806 UV detector at 254 nM. For quantification, peak
areas of dG standards and of three concentration calibration
pure 8-oxodG standards (Sigma) were analyzed during each
HPLC run. A comparison of areas of 8-oxodG standards
injected with and without simultaneous injection of dG
standards ensured that no oxidation of dG occurred during
the HPLC run.
GSA, AASA, CML, CEL, and MDAL measurements in
mitochondrial proteins
GSA, AASA, CML, CEL, and MDAL concentrations were
measured by GC/MS as described previously (25). Briefly,
samples containing 500 g of mitochondrial protein were
delipidated using chloroform:methanol (2:1 v/v) in the pres-
ence of 0.01% butylated hydroxytoluene, and proteins were
precipitated by adding trichloroacetic acid to 10% (v/v) final
concentration, followed by centrifugation. Protein samples
were immediately reduced by overnight incubation with 500
mM NaBH
4
in 0.2 M borate buffer at pH 9.2, and containing
1 drop of hexanol as an antifoam reagent. Protein was
reprecipitated by adding 1 ml of 20% (v/v) trichloroacetic
acid, followed by centrifugation. Isotopically labeled internal
standards ([
2
H
8
]-lysine (CDN Isotopes, Pointe-Claire, Que
-
bec, Canada) were then added; [
2
H
4
]-CML, [
2
H
4
]-CEL, and
[
2
H
8
]-MDAL were prepared as described previously (26 –28);
[
2
H
5
]5-hydroxy-2-aminovaleric acid (for GSA quantification)
and [
2
H
4
]6-hydroxy-2-aminocaproic acid (for AASA quantifi
-
cation) were prepared as described previously (29). The
samples were hydrolyzed at 155°C for 30 min in 1 ml of 6 M
HCl and dried in vacuo. The N,O-trifluoroacetyl methyl ester
derivatives of the protein hydrolysates were prepared as
described previously (26). GC/MS analyses were carried out
on a Hewlett-Packard model 6890 gas chromatograph
equipped with a HP-5MS capillary column (30 m⫻0.25
mM⫻0.25 m) coupled to a Hewlett-Packard model 5973A
mass selective detector. The injection port was maintained at
275°C; the temperature program was 5 min at 110°C, then
2°C/min to 150°C, then 5°C/min to 240°C, then 25°C/min
to 300°C, and finally held at 300°C for 5 min. Quantification
was performed by external standardization using standard
curves constructed from mixtures of deuterated and nondeu-
terated standards. Analyses were carried out by selected
ion-monitoring GC/MS (SIM-GC/MS). The ions used were
lysine and [
2
H
8
]-lysine, m/z 180 and 187, respectively; 5-hy
-
droxy-2-aminovaleric acid and [
2
H
5
]5-hydroxy-2-aminovaleric
acid (stable derivatives of GSA), m/z 280 and 285, respectively;
6-hydroxy-2-aminocaproic acid and [
2
H
4
]6-hydroxy-2-amino
-
caproic acid (stable derivatives of AASA), m/z 294 and 298,
respectively; CML and [
2
H
4
]-CML, m/z 392 and 396, respec
-
tively; CEL and [
2
H
4
]-CEL, m/z 379 and 383, respectively; and
MDAL and [
2
H
8
]-MDAL, m/z 474 and 482, respectively. The
amounts of product were expressed as the molar ratio of
GSA, AASA, CML, CEL, or MDAL/mol lysine.
Mitochondrial complex I and IV
The concentration of mitochondrial complexes I and IV was
estimated using Western blot analysis. Immunodetection was
performed using a monoclonal antibody specific for the
NDUFA9 subunit of complex I and subunit I of complex IV
(1:500 /1:2,000, Molecular Probes, Invitrogen Ltd., Abing-
don, Oxon, UK). An antibody to porin (1:1,000, Molecular
Probes, Invitrogen Ltd.) as a control for total mitochondrial
mass was also used to determine the proportion of complex I
and IV referred to total mitochondrial mass. Peroxidase-
coupled secondary antibodies were used from the Tropix
chemiluminescence kit (Bedford, MA, USA). Signal quantifi-
cation and recording was performed with a CCD camera-
based system (Lumi-Imager) from Boehringer Mannheim
(Mannheim, Germany).
Fatty acid analysis
Fatty acyl groups were analyzed as described previously (25).
Tissue lipids were extracted from homogenate fractions with
chloroform:methanol (2:1, v/v) in the presence of 0.01%
(w/v) butylated hydroxytoluene. The chloroform phase was
evaporated under nitrogen and the fatty acyl groups were
transesterified by incubation in 2.5 ml of 5% (v/v) metha-
nolic HCl for 90 min at 75°C. The resulting fatty acid methyl
esters were extracted by adding 1 ml of saturated NaCl
solution and 2.5 ml of n-pentane. The n-pentane phase was
separated and evaporated under nitrogen. The residue was
dissolved in 75 l of carbon disulfide and 1 l was used for
GC/MS analysis. Separation was performed in a SP2330
capillary column (30 m⫻0.25 mM⫻0.20 m) in a Hewlett
Packard 6890 Series II gas chromatograph. A Hewlett Packard
5973A mass spectrometer was used as detector in the elec-
tron-impact mode. The injection port was maintained at
220°C and the detector at 250°C; the temperature program
was 2 min at 100°C, then 10°C/min to 200°C, then 5°C/min
to 240°C, and finally held at 240°C for 10 min. Identification
of fatty acyl methyl esters was made by comparison with
authentic standards and on the basis of mass spectra. Results
are expressed as mol%. The following fatty acyl indices were
also calculated: saturated fatty acids (SFA): unsaturated fatty
1066 Vol. 20 June 2006 SANZ ET AL.The FASEB Journal
acids (UFA); monounsaturated fatty acids (MUFA); polyun-
saturated fatty acids from n-3 and n-6 series (PUFAn-3 and
PUFAn-6); average chain length (ACL) ⫽ [(⌺%Total14⫻14)
⫹ (⌺%Total16⫻16) ⫹ (⌺%Total18⫻18) ⫹ (⌺%Total20⫻20)
⫹ (⌺%Total22⫻22)]/100; double bond index (DBI) ⫽
[(⌺mol% monoenoic) ⫹ (2⫻⌺mol% dienoic) ⫹ (3⫻⌺mol%
trienoic) ⫹ (4⫻⌺mol% tetraenoic) ⫹ (5⫻⌺mol% pentae-
noic) ⫹ (6⫻⌺mol% hexaenoic)], and peroxidizability index
(PI) ⫽ [(0.025⫻⌺mol% monoenoic) ⫹ (⌺mol% dienoic) ⫹
(2⫻⌺mol% trienoic) ⫹ (4⫻⌺mol% tetraenoic) ⫹
(6⫻⌺mol% pentaenoic) ⫹ (8⫻⌺mol% hexaenoic)].
Statistics
Data were analyzed by Student’s t tests. The minimum con-
centration of statistical significance was set at P ⬍ 0.05 in all
the analyses.
RESULTS
Methionine restriction significantly decreased the rate
of H
2
O
2
production of heart mitochondria with pyru
-
vate/malate as substrates to values 64% those of control
animals (Fig. 1, upper panel). With succinate as sub-
strate no significant differences between control and
MetR heart mitochondria were observed (Fig. 1, upper
panel). However, when the experiments with succinate
as substrate were repeated in the absence of rotenone,
the rate of H
2
O
2
generation of the MetR group was
significantly lower than that of controls.
In liver mitochondria, methionine restriction also
significantly decreased the rate of H
2
O
2
generation to
46% and 43% of control values with pyruvate/malate
and with glutamate/malate as substrates, respectively
(Fig. 1, lower panel). In the presence of succinate,
hepatic mitochondrial H
2
O
2
production also was signif
-
icantly lower in MetR than in control mitochondria.
Maximum rates of H
2
O
2
generation were assayed
using appropriate combinations of substrates and in-
hibitors of the respiratory chain (Table 1). The rate of
H
2
O
2
production of heart mitochondria was signifi
-
cantly decreased by methionine restriction in the pres-
ence of pyruvate/malate⫹rotenone (full reduction of
complex I) but not in the presence of succinate⫹
antimycin A (full reduction of complex III). In the case
of liver mitochondria, no significant differences in
H
2
O
2
generation between control and MetR groups
were observed with pyruvate/malate⫹rotenone, gluta-
mate/malate⫹rotenone or succinate⫹antimycin A
(Table 1).
The rate of O
2
consumption of heart mitochondria
was significantly increased by MetR with pyruvate/
malate in state 3 but not in state 4 (Table 2). In the
presence of succinate, nonsignificant trends to increase
heart mitochondrial O
2
consumption were found that
showed marginal significance (P ⬍ 0.07 in state 4 and
P ⬍ 0.08 in state 3).
In liver mitochondria, the rate of O
2
consumption
was significantly increased by MetR with glutamate/
malate and with succinate in both state 4 and state 3, as
well as with pyruvate/malate in state 3 (Table 2). Only
with pyruvate/malate in state 4 did the increase in O
2
consumption fail to reach statistical significance.
The percentage of electrons in the respiratory chain
directed to ROS generation (the free radical leak, FRL)
in heart mitochondria was significantly decreased by
MetR with pyruvate/malate (to 43% of controls; Fig. 2)
but not with succinate (results not shown). In liver
mitochondria, MetR significantly decreased the FRL
both with the complex I (pyruvate/malate and gluta-
mate/malate) and the complex II (succinate) linked
substrates used to values 28 –39% those of controls
(Fig. 2).
Methionine restriction significantly decreased the
concentration of complex I and complex IV in both
heart and liver mitochondria (Fig. 3).
Figure 1. Rates of H
2
O
2
production of heart and liver
mitochondria from control and methionine-restricted rats.
PYR ⫽ pyruvate/malate; SUCC ⫽ succinate; SUCC-ROT ⫽
succinate without rotenone; GLU ⫽ glutamate/malate;
MetR ⫽ methionine-restricted. Control values in heart mito-
chondria: 0.25 ⫾ 0.03 (PYR); 0.77 ⫾ 0.20 (SUCC); 7.24 ⫾
1.00 (SUCC-ROT). Control values in liver mitochondria:
0.13 ⫾ 0.03 (PYR); 0.93 ⫾ 0.17 (GLU); 0.460 ⫾ 0.04 (SUCC).
Values are means ⫾ se from 6 –8 different animals. Asterisks
represent statistically significant differences between control
and methionine-restricted animals (*P⬍0.05; **P⬍0.01).
1067METHIONINE RESTRICTION AND ROS GENERATION
The levels of the oxidative damage marker 8-oxodG
in mtDNA were significantly decreased by MetR to 48%
of controls in liver and to 65% of controls in heart
(Fig. 4).
All the protein markers of oxidative, glycoxidative,
and lipoxidative damage were significantly decreased
by MetR in both liver and heart mitochondria (Table
3). In heart mitochondrial proteins GSA, AASA, CEL,
and CML were decreased to 67–82% of control values,
whereas in the case of MDAL the values found in MetR
were 50% those of controls (Table 3). In liver mito-
chondria, GSA, CEL, and CML were decreased by MetR
to 70 – 85% of controls whereas AASA and MDAL were
lowered to 68% and 63% of controls, respectively.
Methionine restriction altered the fatty acid compo-
sition of liver (Table 4) and heart (Table 5) mitochon-
dria, so that the total number of double bonds (DBI)
was significantly decreased in both cases. In both kinds
of mitochondria, the fatty acids mainly responsible for
the decrease in DBI were the same. Thus, methionine
restriction significantly increased the fatty acids with no
or a small number of double bonds 18:0, 18:1n-9, and
18:2n-6 whereas it significantly decreased the main
highly unsaturated ones 20:4n-6 and 22:6n-3 (Tables 4
and 5).
Figure 2. Free radical leak (%) of heart and liver mitochon-
dria from control and methionine-restricted rats. The FRL is
the percentage of the total electron electron flow in the
respiratory chain directed to oxygen radical generation (see
Materials and Methods). PYR, pyruvate/malate; SUCC, succi-
nate; GLU, glutamate/malate; MetR, methionine-restricted.
Values are means ⫾ se from 7– 8 different animals. Asterisks
represent statistically significant differences between control
and methionine-restricted animals (*P⬍0.05; **P⬍0.01).
Figure 3. Concentration of protein complexes I and IV in
heart and liver mitochondria from control and methionine-
restricted rats. Values are means ⫾ se from 4 –5 different
animals. Asterisks represent statistically significant differences
between control and methionine-restricted animals
(P⬍0.05).
TABLE 2. Oxygen consumption of heart and liver mitochondria
from control and methionine-restricted rats
a
Control Methionine restricted
Heart mitochondria:
Pyr (E4) 26.2 ⫾ 4.8 33.7⫾5.0
Pyr (E3) 123 ⫾ 16 185 ⫾ 3*
Succ (E4) 86 ⫾ 18 125 ⫾ 15
a
Succ (E3) 137 ⫾ 30 218 ⫾ 48
b
Liver mitochondria:
Pyr (E4) 5 ⫾ 1.2 6 ⫾ 1.0
Pyr (E3) 15 ⫾ 1.5 20 ⫾ 2.3*
Glu (E4) 7 ⫾ 1.2 10 ⫾ 1.2*
Glu (E3) 59 ⫾ 7.4 93 ⫾ 6.8**
Succ (E4) 17 ⫾ 2.0 30 ⫾ 2.9**
Succ (E3) 88 ⫾ 10.2 129 ⫾ 9.4**
a
Pyr, pyruvate/malate; Glu, Glutamate/malate; Succ, succinate;
E4, state 4 (substrate alone); E3, state 3 (substrate⫹ADP). Values are
means ⫾ se from 7–8 different animals and are expressed in
nanomoles of O
2
/min. mg mitochondrial protein. Asterisks repre
-
sent statistically significant differences between control and methi-
onine-restricted groups (*P⬍0.05; **P⬍0.01);
a
(P⬍0.07);
b
(P⬍0.08).
TABLE 1. Maximum H
2
O
2
production in the presence of
respiratory chain inhibitors in heart and liver mitochondria from
control and methionine-restricted rats
a
Control Methionine restricted
Heart:
Pyr⫹Rot 3.53 ⫾ 0.32 2.73 ⫾ 0.17*
Succ⫹AA 23.17 ⫾ 3.40 19.54 ⫾ 2.59
Liver:
Pyr⫹Rot 0.56 ⫾ 0.07 0.69 ⫾ 0.06
Glu⫹Rot 0.82 ⫾ 0.09 0.74 ⫾ 0.08
Succ⫹AA 3.21 ⫾ 0.33 3.39 ⫾ 0.34
a
Pyr, pyruvate/malate; Succ, succinate; Rot, rotenone; AA, anti
-
mycin A. Values are means ⫾ se from 7– 8 different animals and are
expressed in mol of H
2
O
2
/min. mg mitochondrial protein.
*(P⬍0.05 between control and methionine-restricted groups).
1068 Vol. 20 June 2006 SANZ ET AL.The FASEB Journal
DISCUSSION
In this work it is shown for the first time that methio-
nine restriction decreases mitochondrial ROS genera-
tion and oxidative damage to mitochondrial DNA and
proteins. This strongly suggests that the decrease in
methionine intake is the cause of these effects consis-
tently found in many investigations on caloric restric-
tion in rodents.
Caloric restriction is the better known experimental
manipulation that decreases aging rate, but its mecha-
nisms of action are unknown. Many studies have con-
sistently shown that CR decreases mitROS generation
(3), suggesting this can be one of the main mecha-
nisms. But the dietary component responsible for this
change was unknown. A recent study from our labora-
tory found that protein restriction (PR) without strong
caloric restriction also decreases mitROS generation at
complex I, lowers FRL, and decreases 8-oxodG in
mtDNA (8) in rat liver mitochondria in exactly the
same quantitative and qualitative way as CR, whereas
neither lipid restriction (7) nor carbohydrate restric-
tion (unpublished results) modify the rate of mitROS
production. Looking for the protein component re-
sponsible for these effects, we focused on methionine
in the present investigation since various sources of
data currently relate methionine to aging. First, it is
known that MetR without energy restriction increases
maximum life span in rats and mice (14,15), similar to
what was found in PR (10 –13) although the PR effect
on life span is smaller than that of CR. Recent investi-
gations suggest that the increase in maximum longevity
induced by MetR always occurs irrespective of the
particular rat strain selected (30). Second, it has been
found that the protein methionine content is inversely
Figure 4. Oxidative damage to mitochondrial DNA (8-
oxodG) in heart and liver from control and methionine-
restricted rats. MetR, methionine-restricted. Values are
means ⫾ se from 6–7 different animals. Asterisks represent
statistically significant differences between control and methi-
onine-restricted animals (**P⬍0.01).
TABLE 3. Protein markers of oxidative, glycoxidative, and lipoxidative damage in liver and heart mitochondria from control and
methionine-restricted rats
a
Liver mitochondria Heart mitochondria
Control MetR Control MetR
GSA 4193 ⫾ 96 3168 ⫾ 137*** 5004 ⫾ 121 3950 ⫾ 131***
AASA 137 ⫾ 793⫾ 2*** 212 ⫾ 13 143 ⫾ 23*
CEL 369 ⫾ 8 258 ⫾ 10*** 572 ⫾ 22 409 ⫾ 25**
CML 1201 ⫾ 17 1021 ⫾ 30*** 1768 ⫾ 35 1441 ⫾ 43***
MDAL 387 ⫾ 14 245 ⫾ 8*** 452 ⫾ 17 228 ⫾ 14***
a
GSA, glutamic semialdehyde; AASA, aminoadipic semialdehyde; CEL, carboxyethyl-lysine; CML, carboxymethyl-lysine; MDAL, malondi
-
aldehyde-lysine. MetR, methionine-restricted. Values are means ⫾ se from 5–7 different animals and are expressed as mol/mol lysine. Asterisks
represent statistically significant differences between control and methionine-restricted groups (*P⬍0.05; **P⬍ 0.01; ***P⬍0.001).
TABLE 4. Fatty acid analysis of liver mitochondria from control
and methionine-restricted rats
a
Liver mitochondria
Control Methionine restriction P
14:0 0.23 ⫾ 0.003 0.21 ⫾ 0.01 0.224
16:0 16.11 ⫾ 0.31 15.04 ⫾ 0.24 0.022
16:1n-7 0.75 ⫾ 0.09 0.48 ⫾ 0.04 0.022
18:0 18.14 ⫾ 0.22 20.48 ⫾ 0.31 0.000
18:1n-9 7.13 ⫾ 0.40 8.73 ⫾ 0.57 0.064
18:2n-6 20.20 ⫾ 0.49 21.93 ⫾ 0.46 0.031
18:3n-3 0.28 ⫾ 0.01 0.26 ⫾ 0.01 0.368
20:3n-6 0.42 ⫾ 0.07 0.30 ⫾ 0.03 0.139
20:4n-6 28.05 ⫾ 0.44 25.23 ⫾ 0.29 0.000
22:4n-6 1.08 ⫾ 0.12 1.01 ⫾ 0.07 0.623
22:5n-6 0.55 ⫾ 0.04 0.46 ⫾ 0.03 0.120
22:5n-3 0.64 ⫾ 0.04 0.56 ⫾ 0.02 0.124
22:6n-3 6.36 ⫾ 0.16 5.25 ⫾ 0.11 0.000
ACL 18.56 ⫾ 0.01 18.48 ⫾ 0.01 0.000
SFA 34.48 ⫾ 0.13 35.73 ⫾ 0.28 0.006
UFA 65.51 ⫾ 0.13 64.26 ⫾ 0.28 0.006
MUFA 7.88 ⫾ 0.37 9.22 ⫾ 0.57 0.109
PUFA 57.62 ⫾ 0.39 55.03 ⫾ 0.57 0.007
PUFAn-3 7.29 ⫾ 0.17 6.08 ⫾ 0.11 0.000
PUFAn-6 50.32 ⫾ 0.29 48.94 ⫾ 0.61 0.107
DBI 211.17 ⫾ 1.37 196.48 ⫾ 1.14 0.000
PI 196.48 ⫾ 1.94 176.54 ⫾ 1.64 0.000
a
ACL, acyl chain length; SFA, saturated fatty acids; UFA, unsat
-
urated fatty acids; MUFA, monounsaturated fatty acids; PUFA, poly-
unsaturated fatty acids; DBI, double bond index; PI, poliunsaturation
index. Values are means ⫾ se from 5–7 different animals and are
expressed as mol%.
1069METHIONINE RESTRICTION AND ROS GENERATION
related to maximum life span in mammals (16), which
makes sense since methionine is among the protein
amino acids most susceptible to oxidation by ROS (17).
Third, methionine dietary supplementation damages
many vital organs (like cardiovascular system and liver)
and increases oxidative stress (31,32), and similar neg-
ative effects have been found in rats fed high protein
diets (31,33). Fourth, knocking out methionine sulfox-
ide reductase-A (an enzyme that reduces methionine
sulfoxide back to methionine through a thioredoxin-
dependent reaction) lowers maximum longevity and
increases protein carbonyls in mice (17), whereas over-
expression of this enzyme in Drosophila increases its life
span and delays aging (18). Fifth, overexpression of
thioredoxin increases longevity in mice (19). Sixth,
long-lived mutant Ames dwarf mice seem to have
altered methionine metabolism (34).
In agreement with our hypothesis, we found in this
investigation that MetR, using the same dietary proto-
col that increases maximun longevity in rats (14), also
decreases mitROS production, FRL, and oxidative dam-
age to mtDNA, similar to what has been observed in PR
and CR (3,4,20). The decrease in mitROS production
occurred in both liver and heart mitochondria and was
quantitatively strong. In the case of heart mitochondria,
the decrease in ROS generation occurred only at
complex I since it occurred with pyruvate/malate but
not with succinate (⫹rotenone) as substrate, and it also
took place when the assay with succinate was performed
in the absence of rotenone (which allows backward flow
of electrons from succinate to the complex I ROS
generator). In heart mitochondria it has been found
that the decrease in ROS generation in CR also occurs
exclusively at complex I (4). In the case of liver
mitochondria, MetR decreased ROS generation both at
complex I and complex III, since the decreases oc-
curred with the complex II-linked substrate (succinate)
as well as with the complex I-linked ones (pyruvate/
malate and glutamate/malate). In 40% CR, decreases
in ROS generation have been detected only at complex
I in rat liver mitochondria (20).
Concerning the mechanism responsible for the de-
crease in mitROS production during MetR, there are
various possibilities. A simple mechanism can be a
decrease in the concentration of the respiratory com-
plex/es responsible for ROS generation. This has been
described in relation to aging in comparisons between
rats and pigeons, in which the lower rate of mitROS
production of the long-lived species (the pigeon) has
been attributed to its lower complex I content (35).
This could also be a mechanism during food restric-
tion, since variations in the levels of mitochondrial
protein complexes have been described as a result of
CR (36). To test this, we measured the concentration of
complex I and IV in control and MetR animals. Com-
plex I contains the ROS generator site mainly related to
aging, whereas complex IV is known not to produce
ROS and was also measured as a control. Similar results
were found in both heart and liver mitochondria. MetR
decreased the concentration of complexes I and IV.
This strong decrease in complex I content in MetR, as
in the pigeon vs. rat comparison, can contribute to the
decrease observed in mitROS production.
Even if the decrease in complex I helps to decrease
ROS generation, it cannot be the only explanation of
this change for various reasons. First, while ROS pro-
duction with substrate alone decreases in MetR, when
maximum rates of ROS production were assayed with
appropriate combinations of substrate plus inhibitor
(pyruvate/malate plus rotenone and succinate plus
antimycin A), in almost every case the difference in
ROS generation between control and MetR mitochon-
dria disappeared. With substrate alone the respiratory
chain is only partially reduced, so that ROS production
depends both on the amount of ROS generators
present as well as on the degree of electronic reduction
of these generators (the higher their degree of reduc-
tion, the higher will be their rate of ROS production).
But in the presence of the inhibitors, the ROS genera-
tors are fully reduced, so that the degree of reduction
of the generators is no longer a variable. If the decrease
in ROS production observed in MetR were only due to
decreases in the concentration of ROS generators, a
lower ROS production would be observed in the pres-
ence of those substrate⫹inhibitor combinations in
MetR than in the controls. Since this was not the case,
it seems that the degree of reduction of the ROS
generators is involved in the decreases in ROS produc-
tion observed in MetR with substrate alone.
That intrinsic changes at the concentration of the
ROS generators occur in MetR is also indicated by the
TABLE 5. Fatty acid analysis of heart mitochondria from control
and methionine-restricted rats
a
Heart mitochondria
Control Methionine restriction P
14:0 0.21 ⫾ 0.01 0.18 ⫾ 0.002 0.037
16:0 11.77 ⫾ 0.06 11.05 ⫾ 0.11 0.001
16:1n-7 0.29 ⫾ 0.03 0.20 ⫾ 0.02 0.063
18:0 23.30 ⫾ 0.30 24.59 ⫾ 0.36 0.029
18:1n-9 6.10 ⫾ 0.03 7.24 ⫾ 0.22 0.001
18:2n-6 16.69 ⫾ 0.42 18.37 ⫾ 0.19 0.014
20:4n-6 27.88 ⫾ 0.14 26.18 ⫾ 0.40 0.003
22:4n-6 2.85 ⫾ 0.07 2.15 ⫾ 0.15 0.003
22:5n-6 1.31 ⫾ 0.08 1.29 ⫾ 0.04 0.890
22:5n-3 1.43 ⫾ 0.04 1.32 ⫾ 0.02 0.064
22:6n-3 8.11 ⫾ 0.05 7.39 ⫾ 0.22 0.009
ACL 18.85 ⫾ 0.007 18.77 ⫾ 0.006 0.000
SFA 35.29 ⫾ 0.30 35.83 ⫾ 0.35 0.283
UFA 64.70 ⫾ 0.30 64.16 ⫾ 0.35 0.283
MUFA 6.39 ⫾ 0.06 7.44 ⫾ 0.24 0.002
PUFA 58.30 ⫾ 0.33 56.71 ⫾ 0.14 0.005
PUFAn-3 9.55 ⫾ 0.07 8.71 ⫾ 0.24 0.009
PUFAn-6 48.75 ⫾ 0.38 48.00 ⫾ 0.12 0.140
DBI 225.21 ⫾ 0.74 214.97 ⫾ 1.08 0.000
PI 221.26 ⫾ 0.93 206.74 ⫾ 1.24 0.000
a
ACL, acyl chain length; SFA, saturated fatty acids; UFA, unsat
-
urated fatty acids; MUFA, monounsaturated fatty acids; PUFA, poly-
unsaturated fatty acids; DBI, double bond index; PI, poliunsaturation
index. Values are means ⫾ se from 5–7 different animals and are
expressed as mol%.
1070 Vol. 20 June 2006 SANZ ET AL.The FASEB Journal
strong decrease in percent free radical leak (FRL)
observed in MetR. MetR mitochondria not only pro-
duce less ROS per unit time, but also produce less ROS
per unit elecron flow in the respiratory chain, similar to
what has been observed in CR (4,20) and PR (8) rats as
well as in long-lived vs. short-lived animal species (36).
In all these models of slow aging, the mitochondria are
more efficient since they have a lower rate of ROS
generation without the need to decrease oxygen con-
sumption (and thus energy production), an interesting
capability. In the case of MetR, not only was the
consumption of oxygen not decreased, but it was even
increased, providing more clues concerning the mech-
anisms of decrease in ROS generation. Contrary to
intuitive thinking, when in the same individual the rate
of mitochondrial O
2
consumption increases it tends to
decrease (instead of increase) ROS generation due to
two different effects. On the one hand, the degree of
reduction of the respiratory chain decreases, which
tends to decrease ROS generation. On the other hand,
the increased oxygen consumption locally lowers the
pO
2
, and this lowers ROS production since the K
M
of
the ROS generators (which have low affinity for O
2
)
falls within the range of physiological tissue pO
2
, mean
-
ing that ROS production is pO
2
dependent at physio
-
logical tissue oxygen partial pressures. For these two
reasons, the increase in oxygen consumption of the
MetR mitochondria helps lower their rate of ROS
production while decreasing their FRL (increase in
efficiency in avoiding ROS generation).
Another possibility is that the decrease in ROS pro-
duction is related to a mild uncoupling induced by the
MetR protocol. This is suggested by the general trend
to increased oxygen consumption of the MetR mito-
chondria, which suggests that the proton gradient was
lowered by MetR. This would be relevant since it is
known that high proton gradients raise ROS produc-
tion at least at complex III. In any case, such changes in
oxygen consumption do not occur in 40% CR and 40%
PR (4,8,20). The reason for this difference could be the
particular MetR protocol used in our investigation:
methionine was restricted by 80% instead of 40% (as in
the previous CR and PR studies), and it was substituted
for glutamate in the diet. More studies are needed to
ascertain whether the decrease in ROS production still
occurs and the increases in oxygen consumption disap-
pear when MetR is performed at 40% and without
substituting it for dietary glutamate.
Another mechanism can also be involved in the
decrease in mitROS production in MetR. It has been
reported that addition of oxidized glutathione to mito-
chondrial complex I increases its rate of superoxide
radical generation (37), and the same seems to occur
after addition of homocysteine to rat heart mitochon-
dria (38), suggesting that MitROS production can be
regulated by thiol agents. Toxic effects of dietary me-
thionine have been related to its conversion to and
increase in homocysteine levels (31,39). Homocysteine
has a free thiol group that can react with protein thiols,
leading to protein mixed disulfides. Thus, MetR could
decrease ROS generation through decreases in homo-
cysteine, which would decrease thyolization of complex
I. Homocysteine levels also increase with age in humans
and represent a risk factor for aging and free radical-
associated degenerative diseases (40).
Concerning oxidative damage, in this investigation it
was found that MetR decreases 8-oxodG levels in
mtDNA in both liver and heart. This also occurs in the
same rat organs after both CR (4,20) and PR (8). This
further supports the idea that the decrease in methio-
nine ingestion is responsible for the decrease in mito-
chondrial oxidative stress observed in CR and PR. The
decrease in 8-oxodG in MetR also agrees with the lower
mitROS generation observed in this dietary manipula-
tion, similar to what has been found in CR (4,20) and
PR (8). Similar reasoning can be applied to the strong
decreases in all of the five different markers of oxida-
tive damage to mitochondrial proteins measured in this
study. Decreases in GSA, MDAL, CML, and CEL have
been observed in heart mitochondrial proteins of CR
rats (5,6). Of these protein markers, some of them
(CML and MDAL) are dependent on lipid peroxida-
tion. Since lipid peroxidation increases strongly as a
function of the number of double bonds per fatty acid,
we measured the full fatty acid composition of heart
and liver mitochondria. We found that in both tissues
MetR significantly decreases the total number of dou-
ble bonds. Thus, part of the decrease in CML and
MDAL can be secondary to this decrease in the number
of fatty acid double bonds. But this cannot be the full
explanation because other protein markers, not depen-
dent on lipid peroxidation (GSA, AASA and CEL),
were also decreased by MetR, and because the quanti-
tative decrease in double bonds was relatively small
compared to that in MDAL and CML. Thus, it seems
that the decrease in mitROS generation induced by
MetR leads to a generalized decrease in oxidation,
glycoxidation, and lipoxidation of mitochondrial pro-
teins.
Concerning the particular fatty acid modified by
MetR, the most important changes observed (quantita-
tively) were the same in both tissues. MetR decreased
the highly unsaturated fatty acids 20:4n-6 and 22:6n-3,
and substituted them for the much less unsaturated
18:2n-6, 18:1n-9, and 18:0. It is striking that this is
essentially the kind of difference that has been ob-
served when comparing animals with different longevi-
ties. Long-lived animals have less unsaturated mem-
branes in tissues and mitochondria than short-lived
ones, mainly due to a decrease in 22:6n-3 and 20:4n-6
and to an increase in 18:2n-6, leading to a lower lipid
peroxidation and lipoxidation-dependent damage to
macromolecules (41).
In summary, it is shown here for the first time that
methionine restriction, similar to both caloric and
protein restriction, decreases mitochondrial ROS gen-
eration, increases the efficiency of the respiratory chain
in avoiding electron leak to oxygen, and lowers steady-
state oxidative damage to mitochondrial DNA and
proteins. This suggests that the decrease in methionine
1071METHIONINE RESTRICTION AND ROS GENERATION
ingestion can be the single molecular component re-
sponsible for the decrease in mitochondrial ROS gen-
eration and oxidative stress that occurs during caloric
restriction, and thus for part of the decrease in aging
rate elicited by this dietary manipulation, although a
role for other dietary amino acids cannot be discarded
without further investigation.
This study was supported in part by I⫹D grants from the
Spanish Ministry of Science and Technology (BFI2003–
01287), the Generalitat of Catalunya (Departament
d’Universitats, Recerca, i Societat de la Informacio´, DURSI,
ref. 2005SGR00101), and the Spanish Ministry of Health (FIS
02–0891, 04 – 0355, and 05–2241) to R.P., from Fundacio la
Caixa to R.P. and M.P.O, and from the Spanish Ministry of
Science and Education (BFU2005– 02584) to G.B. We thank
David Argiles for excellent technical assistance. P. Caro and
A. Sanz received a predoctoral fellowship from the Ministry of
Science and Technology and from the Complutense Univer-
sity, respectively.
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Received for publication December 12, 2005.
Accepted for publication January 24, 2006.
1073METHIONINE RESTRICTION AND ROS GENERATION
